Clostridium difficile is a Gram-positive spore forming organism that is a major cause of antibiotic-associated diarrhea (AAD) and pseudomembranous colitis (PMC). C. difficile was first isolated from the feces of patients undergoing clindamycin treatment in 1978 and identified as the pathogenic organism responsible for PMC (Larson 1978). C. difficile is an emerging pathogen responsible for severe gastrointestinal disease by production of potent toxins that damage the mucosal lining of the colon. Gastrointestinal infections range in severity from colonization to severe diarrhea, PMC, toxic megacolon, colonic perforation and death. The incidence of C. difficile infections in hospitalized patients has increased dramatically in recent years, with current incidence ranging from 2 to 30 cases per 1000 hospital admissions (Barbut, 2000, Johnson, 1990, Bartlett, 1992).
Conservative estimates place the cost of this disease in the U.S. alone at more than $1.1 billion per year. C. difficile infection increases hospital length of the stay by an average of 8 days and in geriatric patients by 36 days. An increasing proportion of patients present with life-threatening fulminant colitis. A recent study identified this incidence to be ˜1.6% to 3.2% overall. Patients undergoing colectomy for C. difficile colitis had a mortality rate of 57%. (Dallal, 2002). be ˜1.6% to 3.2% overall. Patients undergoing colectomy for C. difficile colitis had a mortality rate of 57%. (Dallal, 2002).
Current diagnosis is based on analysis of fresh diarrheic stools. The gold-standard is the cell-culture cytotoxin test, which detects toxin B by its effects on a cultured monolayer of human fibroblasts (Chang, 1979). A frequently used alternative is an antibody-based enzyme immunoassay test that directly identifies toxin A or both toxin A and B (Laughon, 1984). Endoscopic diagnosis of pseudomembranous colitis by the presence of characteristic white-yellowish plaques in the colon is a highly specific sign and pathognomonic of the disease (Fekety, 1993, Kelly, 1998).
Once diagnosed, conventional therapy involves reducing the predisposing conditions, (e.g., withdrawing the inciting antimicrobial therapy if possible), supportive therapy with intravenous fluids, and directed antimicrobial therapy with oral metronidazole (500 mg three times daily or 250 mg every 6 hrs) or oral vancomycin (125 mg every 6 hr). A typical duration of therapy is 10-14 days. Failure to respond (3-10%) may reflect the presence of ileitis or toxic megacolon, with both conditions preventing the drugs from reaching sufficient levels in the colon lumen and can require emergency surgical colostomy. Relapse following treatment is also common, occurring in 20-25% of cases (Mylonakis, 2001, McFarland, 1994). C. difficile infection can be a particular problem in immunodeppressed patients (e.g., transplant recipients and human immunodeficiency virus (HIV)-infected patients) who have higher rates of infection and significantly higher relapse rates. An emerging problem is the development of C. difficile isolates resistant to metronidazole and with intermediate resistance to vancomycin (Pelaez, 2002).
Most cases of C. difficile infection are acquired during hospitalization (nosocomial), although community-acquired cases are increasingly being reported (Hirschhorn, 1994, Kyne, 1998). As a spore-forming organism, C. difficile can persist as an infectious organism in a form that highly resistant to the action of disinfectants and can be recovered from almost every surface in the room of patients with C. difficile infection (Brazier, 1998, Nath, 1994). C. difficile spores are resistant to alcohol solutions routinely used for hand hygiene by health care providers. The ubiquitous presence of C. difficile spores is illustrated clearly by the fact that ˜40% of the clinical recurrences are actually re-infections by a different strain (Alonso, 2001). C. difficile has also emerged as a significant pathogen of farm animals; causing an often-fatal hemorrhagic enteritis in foals, a nosocomial, antibiotic-associated disease in adult horses. Enteric disease is now recognized in ostriches, neonatal pigs, companion animals and calves. (Songer, 2004).
Clostridium difficile colonizes the colon of patients undergoing antibiotic therapy and produces two toxins responsible for the disease. These two toxins, TcdA and TcdB, are encoded on a pathogenicity locus. A critical role of toxin as the determinant of disease is supported by evidence: i) non-toxigenic strains are non-pathologic, ii) development of a strong antibody response reduces recurrence, and iii) the emerging clinical efficacy of toxin-binding resins.
Well-identified risk factors (e.g., use of an antimicrobial agent, advanced age, previous surgery and immunodepression, including transplantation and human immunodeficiency virus infection) underscore the need for novel agents or strategies directed toward prevention of primary infection/disease and post-treatment recurrence particularly attractive.
The clinical disease is believed to be primarily due to Toxin A, a tissue damaging enterotoxin. Toxin B is known to be a potent cytotoxin. Some evidence suggests that Toxins A and B act synergistically, with the initial tissue damage caused by Toxin A being necessary for Toxin B to exert its toxic effects.
Therefore, although Toxin A is believed to cause most of the clinical symptoms, Toxin B may also play an important role in the disease. C. difficile is demonstrated in about 20% of antimicrobial-related diarrhea. The etiology of the remaining 80% of cases is still unknown (a potential role of other microorganisms, such as Clostridium perfringens, Staphylococcus aureus or Candida albicans, has been suggested).
Most cases of C. difficile-associated diarrhea (CDAD) are acquired in hospitals, although community-acquired cases are increasingly being reported. The most important risk factor for CDAD is the use of an antimicrobial agent during the previous 18 weeks (90% of cases), even in the form of a single prophylactic dose. Almost any antimicrobial agent may cause CDAD, although the risk is claimed to be especially high after the administration of clindamycin. Other implicated drugs include ampicillin, amoxicillin and cephalosporins, quinalones, and others, including even vancomycin and metronidazole. Other risk factors for the development of CDAD include advanced age, previous surgery and immunodepression, including transplantation and human immunodeficiency virus infection. Accordingly, there is a great need for effective preventative strategies.
Healthy carriers of C. difficile should not receive antibiotic therapy. Drugs commonly used for the therapy of CDAD are metronidazole (oral or intravenous) and vancomycin (only oral or rectal). Indications for therapy are the detection of a toxin-producing C. difficile strain in a patient with disease (diarrhea, fever, leucocytosis or compatible findings on colonoscopy). The key elements of therapy are to reduce the predisposing conditions (withdraw of unnecessary antimicrobials), provide supportive therapy with fluids, avoidance of anti-peristaltic agents, treatment with effective antimicrobial therapy. Oral metronidazole (500 mg TID) or oral vancomycin (125 mg every 6 h) have response rates near 90%. No well-performed studies have established the optimal normal duration of therapy. Although unproven, some authors recommend longer therapy to avoid recurrence. A therapeutic response usually involves the resolution of fever on the first day and of diarrhea on the fourth or fifth day. Metronidazole is frequently preferred to vancomycin because of lower cost, and because it potentially avoids selection for vancomycin-resistant enterococci.
The most common complication of the treatment of CDAD is relapse (occurring in up to 20-25% of cases). Immunodepressed patients (transplant recipients and human immunodeficiency virus-infected patients) have a higher index of relapse. Relapse should be suspected when the symptoms reappear after completion of therapy. The majority of relapses respond to another 10-day course of therapy with the same antimicrobial agent. However, 35% of patients may have subsequent relapses. These cases represent a major clinical problem. The optimal approach, a longer duration of antibiotic therapy, the use of pulses of vancomycin (125 mg/day), or the administration of resins to absorb the toxins (4 g of colestyramine BID), the use of probiotics (Saccharomyces boulardii or Lactobacillus) or the administration of intravenous immunoglobulins have all been proposed. Forty percent of clinical recurrences may actually reflect re-infections by a different strain. This suggests that these patients are exquisitely susceptible to reinfection.
The prevention and control of CDAD include: (i) the judicious use of antibiotics; (ii) contact precautions; and (iii) adequate environmental cleaning. Restriction of antimicrobial usage is a measure of proven efficacy in decreasing the incidence of CDAD. Accordingly the Department of Health and Public Health Laboratory Service Joint Working Party (1994) recommended: (i) written guidelines on correct antimicrobial use; (ii) restriction of susceptibility reports by the microbiology laboratory; (iii) programs of continuous medical education for healthcare workers; (iv) control and restriction of antimicrobial agent prescriptions; (v) automatic dates for termination of therapy; (vi) contact with the microbiologist or infectious disease specialist in special situations; (vii) avoidance of unnecessary use of antimicrobial agents; (viii) avoidance of wide-spectrum antimicrobial agents when feasible; (ix) avoidance of the use of antimicrobial agents especially linked to a high-risk of CDAD; (x) strict control of surgical prophylaxis.
Necessary first steps in the pathogenesis of disease are an antecedent disruption of the normal colonic flora followed by exposure to a toxigenic strain of C. difficile. Mechanistic steps following exposure include: i) ingestion of spores, ii) survival in the GI tract, iii) spore germination in the small bowel, iv) expansion and growth of toxigenic C. difficile, v) effective colonization and competition with commensal organisms, vi) possible adhesion, vii) toxin elaboration, and viii) binding and acting on epithelial cells.
The ability of C. difficile to form spores is thought to be a key feature that enables it to persist in patients and the physical environment for long periods—thereby facilitating hospital transmission. C. difficile is transmitted through the fecal-oral route. Based on animal models, most ingested vegetative cells are killed in the stomach, with only 1% of the inoculum passing into the small bowel. This may explain the association with potent acid suppressive therapies, such as proton pump inhibitors. C. difficile spores are acid resistant and pass through the stomach. Spores may germinate in the small bowel upon exposure to bile acids. A number of virulence factors, including flagellae and hydrolytic enzymes produced by the organism, have been associated with the development of disease. Nevertheless, the best characterized and most important virulence factors are the C. difficile exotoxins, toxins A and B
Only toxigenic strains are associated with the development of C. difficile diarrhea. Toxin A is thought to play a more critical role than toxin B in the pathogenesis of C. difficile diarrhea because only it is associated with extensive tissue damage and fluid accumulation in experimental animal models. Toxin B, on the other hand, lacks direct enterotoxic activity and may play a role only after the gastrointestinal wall has been damaged by toxin A. However, toxin A-negative/toxin B-positive virulent C. difficile strains have been described, demonstrating that toxin A is not absolutely essential for virulence.
The key involvement of toxin A in most cases of disease have led to strategies designed to bind toxin released in the gut before it can interact with the epithelial receptor. A phase 2 study of the toxin-binding polymer tolevamer in patients with C. difficile associated diarrhea was presented demonstrating that tolevamer and vancomycin have similar efficacy in the treatment of mild-moderate CDAD. This randomized, double-blind, double-dummy, active-controlled phase 2 trial compared the safety and efficacy of monotherapy with 1 g or 2 g tolevamer TID versus a standard oral dose of 125 mg vancomycin QID in 289 patients with a first episode or recurrent CDAD. There was no statistically significant difference between vancomycin and 6 g tolevamer in either primary or recurrent CDAD patients. In the per protocol population, the definitive recurrence rate confirmed by a positive toxin assay was 19% with vancomycin and 10% with 6 g tolevamer (not statistically significant (p=0.185). In the per protocol population with recurrent CDAD at enrollment, the definitive recurrence rate was 27% with vancomycin and 0% with 6 g tolevamer (p=0.07). Others have produced and tested an injectable vaccine containing inactivated C. difficile toxins A and B. Four doses of this toxoid vaccine was shown to be immunogenic and well tolerated in a Phase I clinical study. This approach is currently being tested in clinical trials.
Hospitals and long-term care facilities are major reservoirs of C. difficile infection. Infected patients with diarrhea release C. difficile organisms and microscopic spores that persist in the environment for years. There is currently no effective method to prevent the transmission of C. difficile spores in healthcare facilities. Consequently, C. difficile outbreaks are frequent and can lead to the temporary closure of hospital units. At most, 8% of adults in the community are colonized with C. difficile. Once hospitalized, the risk of becoming colonized with C. difficile is proportional to the length of stay. Twenty percent of patients will become colonized after two weeks of hospitalization, and up to 50% will be colonized after being hospitalized for four weeks. This increase is presumed to be due to exposure to C. difficile in the hospital environment.
Considerable data demonstrate the potential therapeutic role of binding toxin to prevent interaction with physiologic receptors on the epithelial cell surface. Nilsson et al (Bioconjug Chem. 1997 8(4):466-71) showed the utility of solid supports for toxin neutralization. They immobilized enzymatically synthesized alpha Gal(1-3) beta Gal(1-4)Glc trisaccharide, producing a support that efficiently neutralized Clostridium difficile toxin A. The results from this study show that solid supports have potential to serve as inexpensive therapeutics for bacterial diseases mediated by toxin secreted into the lumen. A follow-up study (Gastroenterology. 1996 August;111(2):433-8) showed that pretreatment of rats by gavage dramatically reduced toxin A-associated fluid secretion and permeability. Braunlin et al (Biophys J. 2004 July;87(1):534-9) demonstrated the utility of tolevamer, (GT160-246), a sodium salt of styrene sulfonate polymer, to inhibit the biological activity of C. difficile toxins.
There is a continuing need in the art to develop treatment protocols and reagents to prevent or ameliorate the effects of C. difficile infection.